[ 23 ]
First publ. in: Transactions of the British Mycological Society 64 (1975), 1, pp. 23-28
Trans. Br. mycol. Soc. 64 (I), 23-28 (1975)
Printed in Great Britain
CYTOFLUOROMETRY OF DNA IN UREDOSPORES OF
PUCCINIA GRAMINIS F.SP. TRITICI
By P. G. WILLIAMS* AND K. W. MENDGEN
Institut fur Pflanzenpathologie und Pflanzenschutz, Georg-August Universitat,
Giittingen, Federal Republic of Germany
(With Plate 3 and
2
Text-figures)
The nuclear DNA content of uredospores of a monokaryotic, variant strain of
Puccinia graminis f.sp. tritici was determined using Feulgen fluorescence photometry. The relative values obtained when compared with haploid nuclei in
uredospores of the parent dikaryotic strain, agree with the proposition that the
monokaryotic variant is diploid. Measurements of DNA content also indicated
that each nucleus in the abnormally large, binucleate uredospores formed by
the variant strain, has twice as much DNA as a haploid nucleus.
A study of uredospores with the light microscope indicated that a
monokaryotic, variant strain of Puccinia graminis f.sp. tritici was diploid
(Williams, 1974). The investigation reported here sought to confirm this
finding by measuring the deoxyribose nucleic acid (DNA) content of
uredospore nuclei of the variant strain.
MATERIALS AND METHODS
Rust strains
The monokaryotic strain was obtained from Prof. 1. A. Watson, Department of Agricultural Botany, University of Sydney. It is designated culture
71868 in the University's collection of rust strains. As mentioned elsewhere
(Williams, 1974), this strain was derived from a ur~dium resulting from an
inoculation of wheat with mycelium of the axenic monokaryon VIC
(Maclean, Scott & Tommerup, 1971). Nuclei in uredospores of culture
71868 were compared with haploid nuclei in the parent, dikaryotic strain,
culture 334. Culture 334 uredospores formed the axenic culture that
produced the monokaryotic line VI (Maclean & Scott, 1970). The line VI
was the parent OfVIC (Maclean et al. 1971). The strains were maintained
on wheat seedlings (cv. Nordgau) in separate growth cabinets at 23°.
Feulgen staining
Staining nuclei in mature uredospores is difficult and special treatments
must be used to alter the spore wall and the relative affinity for dyes of
nuclei and cytoplasm (Williams, 1974). Conventional methods are success-
* Permanent address: Department of Agricultural Botany, University of Sydney,
Sydney, 2006.
Konstanzer Online-Publikations-System (KOPS)
URL: http://www.ub.uni-konstanz.de/kops/volltexte/2008/4789/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-47892
24
Transact£ons Brit£sh Mycolog£cal SoC£ety
ful when, after germination, the nuclei have migrated from the spore into
the germ-tube. Samples of uredospores taken from single, isolated uredosori
were allowed to germinate on a drop of water on a microscope slide
previously smeared with egg white. Slides carried one sample each of the
monokaryotic and dikaryotic strain arranged side by side in the centre and
separated by a streak of petroleum jelly. Three to four hours after seeding
the slides were dried and the germinated uredospores were fixed for 30 min
in 6 vo!. 96 % ethyl alcohol, 3 vo!. chloroform, I vo!. acetic acid. After
transfer to water through an alcohol series the preparations were hydrolysed 5 min in 4 N-HCI at 300 (the optimum conditions of hydrolysis were
determined in the experiments described in Results). The slides were
soaked 5 min in S02-water and then immersed for 4 h in the dark in
p-rosaniline-Schiff reagent (5'0 gp-rosaniline (Chroma, Stuttgart)/l 10- 3
N-HCI, reduced overnight with 6'0 g/l sodium metabisulphite, decolorized
with 1'0 g/l Norit, filtered). Excess reagent was removed in four washes of
S02-water and two of distilled water. After drying in air the preparations
were mounted in immersion oil (Leitz) under one coverglass and sealed
with clear nail lacquer. To prevent any bleaching of the stain, the slides
were kept in the dark prior to their measurements. As controls, slides were
prepared in the same manner except that hydrolysis was omitted.
Fluorescent nuclei were photographed with Kodak Tri-X Pan film
developed in EMOFIN (Tetenal, Hamburg).
(e·l
int
109
me
l)JVJt r.neasurer.nent
DNA content of nuclei was determined by measuring the red fluorescence of Feulgen-stained nuclei illuminated with green light (Kasten,
Burton & Glover, 1959). The instrument available to the investigation was
equipped for dark-field illumination. Tbis relatively simple means for
fluorescence excitation proved to be adequate, although incident illumination has been recommended for fluorescence cytophotometry (Bohm &
Sprenger, 1968). Measurements were made with a Leitz MPV I Microscope Photometer with variable diaphragm (Leitz, Wetzlar, Germany)
(Bohm & Sprenger, 1968). The excitation beam, originating from a highpressure Hg lamp, 200 W, passed a heat filter (2 mm KG I, Schott,
Mainz), the excitation filters for green excitation (4 mm BG 38 and Al 546,
both from Schott, Mainz) and the dark-field condenser D 1.20 A to give
a dark-field illumination. For observation, an oil-immersion objective
FI 95 N.A. I. 10 and a barrier filter K 610 (Schott, Mainz) was used. The
measurement was done with a RCA I P 2 I photomultiplier and a microammeter (Norma, Wien). To reduce fading when centring and focusing,
the barrier filter was removed and the object was illuminated with a lowintensity tungsten lamp. For measuring, the barrier filter was put back and
the excitation beam directed to the object with the help of a 45 0 reflecting
mirror. The summit reading of the ammeter was recorded and the excitation cut off again by turning the mirror. Thus, exposure to excitation light
was restricted to a minimum and fluorescence fading became negligible.
As no standard was used, readings of the ammeter are relative and unique
for each experiment. During every experiment, instrument calibration
wa
Sp
ha'
wa
on]
Nu
flu
m
del
COl
fiel
43
va.
th<
ar
cla
m(
nu
fig
Cc
pn
DNA in Puccinia. P. G. Williams and K. W. Mendgen
Ito
80
)n
de
.::-
be
.~ '2 60
!ld
I1g
.e ::;>. 50
In
er
~.~ 40
0-
xe
xe
In
,-3
ed
of
ns
ed
.es
re
m
:5-
n,
as
Dr
11-
&
D-
f)
1-
t,
6,
re
re
le
Dg,
vId
Ig
}It
e.
le
ill
25
70
~.~
1)
u
.~
"'.D
~
i:i:
::l
2-
30
20
2
3
4
5 6 7 8 9 IO II
Hydrolysis time (min)
12 13 14 15 16
Fig. I. Feulgen fluorescence intensity (in arbitrary units) of haploid nuclei in uredospore germ-tubes of culture 334 of Puccinia graminis f.sp. tritici in relation to hydrolysis
time (min) in 4 N-HCI at 30°. Vertical line represents standard deviation.
(e.g. measuring diaphragm) was kept strictly constant. The fluorescence
intensity of a nucleus was obtained from the difference between the reading given with the nucleus in the measuring field and that given by a field
including the adjacent cytoplasm.
RESULTS
Preliminary experiments suggested that hydrolysis in 1 N-HCl at 60°
was unsuitable and hydrolysis with 4 N-HCI at 30° as used by Bbhm &
Sprenger (1968) was adopted. Measurements of fluorescence intensity of
haploid nuclei in germ-tu bes ofculture 334 showed that maximum staining
was obtained after 4-6 min hydrolysis (Fig. 1). Fluorescence of nuclei was
only slightly increased when fixation was increased from 30 min to 16 h.
Nuclei in control (unhydrolysed) preparations gave no measurable
fluorescence, but a small amount of Feulgen reactive material was present
in germ-tube walls before and after hydrolysis.
The stoichiometry of fluorescence intensity and- DNA content was
demonstrated using dikaryons in culture 334. Ammeter readings were
compared when one nucleus or a pair of nuclei were within the measuring
field. Ten haploid nuclei, measured separately, gave a mean value of
43 ± 5; the mean of five dikaryons was 89 ± 10, that is, abou t twice the
value for single nuclei.
Comparisons of nuclear DNA content between the monokaryotic and
the dikaryotic strain were made between the adjacent samples on each of
a number of slides. The data from two slides are shown in Fig. 2. A single
class of values was found in each strain. According to a t test of the sample
means the data fit the hypothesis that the DNA content of culture 71868
nuclei (PI. 3, fig. 1) is double that of haploid nuclei in culture 334 (PI. 3,
fig. 2). (Because of dissimilar variances the modified t test of Cochrane &
Cox (1957) was used for the data from slide 2.) This result agrees with the
previous observations of comparative size of uredospores and their nuclei
26
Transactions British Mycological Society
aCl'
Slide I
-0 8
u
2
<....
6
.......
4
0
Mean=36±7-6
ind
apl
All
an(
difl
Mean=76±6-5
.J:>
E 2
::l
Z
PUt
20
30
40
50
60
70
80
90
100
- tl
DNA content (arbitrary units)
abs
OUI
-.,
u::l
<:
<....
0
.......
oD
E
::l
Z
car
tiOl
Slide 2
~l
(
Mean=77±ll
Mean=35±6-5
obt
dir:
pm
thi
30
20
40
50
60
70
80
90
100
DNA content (arbitrary units).
Fig_ 2. Frequency distribution of DNA content (arbitrary units) of germ-tube nuclei in
dikaryotic (culture 334, open bars) and monokaryotic (culture 71868, shaded bars)
strains of Puccinia graminis f.sp. tritici.
Table I. DNA content in arbitrary units (mean ± standard deviation) of nuclei in
normal, uninucleate and large, binucleate uredospores formed by the monokaryotic
culture 71868 ofP. graminisfsp. triitici
Number of
uredospores
measured
Uninucleate
uredospores
Binucleate
uredospores
8
49± 7'0
4'!±12
(Williams, 1974) indicating that uredial cultures derived from the axenic
monokaryon VI C of Maclean et al. (197 r) are diploid.
The monokaryotic culture 71868 forms small numbers of abnormally
large uredospores, most of which contain two large nuclei (Williams, 1974).
The measurements of fluorescence given in Table I show that each nucleus
in the germ-tube of a binucleate uredospore (PI. 3, fig. 3) has the same
DNA content as the single nucleus in a normal-sized uredospore of this
strain, thus demonstrating that the large uredospores contain two diploid
nuclei.
DISCUSSION
The DNA values of germ-tube nuclei comprised single classes presumably representing I C and 2 Cor 2 C and 4C levels for haploid and diploid
nuclei respectively. No information was obtained to discriminate between
these alternative possibilities. However, a recent investigation of nucleic
an<
of
ma
Ba!
BR~
Wr
D A in Puccinia. P. G. Williams and K. W. Mendgen
27
:mhesis in Uromyees Jabae (Pers.) Wint. sporelings (Staples, J974)
.ndicated that an S phase precedes the nuclear ;division associated with
appressorium formation in this rust fungus (Maheshwari, Hildebrandt &
. Jen, 1967). In Uromyees it appears that the uredospore nuclei are in G1
and remain in that state in the absence of a stimulus leading to the
differentiation of an infection structure. If the same situation holds in
Pueeinia graminis - that is, that germ-tube nuclei are in G 1 of the cell cycle
- the DNA values we observed represent J C and 2 C levels.
The DNA content of fungal nuclei has previously been determined by
absorption photometry of DNA (Bryant & Howard, 1969). The results of
our experiments show that the relative amount of DNA in fungal nuclei
can also be measured by fluorescence photometry using dark-field illumination, a simpler method and one requiring relatively inexpensive equipment.
Our results confirm the work of Williams & Hartley (J 97 I), who first
obtained evidence that Pueeinia graminis f.sp. tritiei can exist as a somatic
diploid. The techniques we have used will be useful in exploring the
possible significance of this discovery in relation to asexual variation in
this pathogen.
We are grateful to the Wheat Industry Research Council of Australia
and the Alexander von Humboldt Foundation for financial support to one
of us (P.G.W.) and to Dr D. J. Maclean for helpful criticism of the
manuscript.
REFERENCES
In
'ie
c
y
I.
5
e
s
i
BOHM, N. & SPRENGER, E. (1968). Fluorescent cytophotometry: A valuable method for
the quantitative detennination of nuclear Feulgen-DNA. Histochemie 16, 100-118.
BRYANT, T. R. & HOWARD, K. L. (1969)' Meiosis in the Oomycetes: 1. A microspectrophotometric analysis of deoxyribonucleic acid in Saprolengnia terrestris. American
Journal of Botany 56, 1075-1083.
COCHRANE, W. G. & Cox, G. M. (1957). Experimental designs, 2nd ed. New York: John
Wiley & Sons.
KASTEN, F. H., BURTON, V. & GLOVER, P. (1959). Fluorescent Schiff-type reagents for
cytochemical detection of polyaldehyde moieties in sections and smears.' Nature,
London 184, 1797-1798.
MACLEAN, D.]. & SCOTT, K.]. (1970). Variant forms of saprophytic mycelium grown
from uredospores of Puccinia graminis f.sp. tritici. Journal of -General Microbiology 64,
19- 2 7.
MAcLEAN, D. ]., SCOTT, K.]. & TOMMERup, 1. C. (1971). A uninucleate wheat-infecting
strain of the stem rust fungus isolated from axenic cultures. Journal if General
Microbiology 65, 339-342.
MAHESHWARI, R., HILDEBRANDT, A. C. & ALLEN, P.]. (1967). The cytology of infection
structure development in uredospore germ tubes in Uromyces phaseoli var. typica
(Pers.) Wint. Canadian Journal of Botany 45, 447-450.
STAPLES, R. C. (1974). Synthesis of DNA during differentiation of bean rust uredospores.
Physiological Plant Pathology (in the Press).
\-YILLIAMS, P. G. (1974). Evidence for diploidy of a monokaryotic strain of Puccinia
graminis f.sp. tritici. Transactions of the British Mycological Society (in the Press).
WILLIAMS, P. G. & HARTLEY, M.]. (1971). Occurrence of diploid lines of Puccinia graminis
tritici in axenic culture. Nature New Biology 229, 181-Il32.
28
Transactions British Mycological Society
EXPLANATION OF PLATE
3
Red fluorescence of Feulgen-stained nuclei in germ-tubes of Puccinia graminis f.sp. tritici
uredospores.
Fig. I. Culture 71868, uninucleate. x 1100.
Fig. 2. Culture 334. x 1100.
Fig. 3. Culture 71868, binucleate. x 1100.
(Accepted for publication 19 June 1974)
Tl
Trans. Br. mycol. Soc.
Vol. 64.
Plate 3
211
11+11
211+211
(Facing
p.
2fl)